Structural and unfolding features of HlyT, a tetrameric LysR type transcription regulator of Vibrio cholerae

Abstract

HlyT from Vibrio cholerae is a positive regulator of Na⁺/H⁺ antiporter, important for the survival of the organism in an aquatic environment and within the human host. Here we report cloning, over-expression and purification of HlyT. Analytical gel filtration and glutaraldehyde cross-linking indicate existence of tetrameric and dimeric forms of HlyT in solution. We propose an unfolding model of HlyT on the basis of guanidine hydrochloride-induced equilibrium unfolding, analyzed by CD and spectrofluorimetric studies. The apparent two-state unfolding pathway of HlyT probably contains at least two cryptic intermediates, one with a more stable or compact structure than the native and another, a molten globule. The tetrameric structure, stabilized by strong hydrophobic interaction between the subunits, is retained even in the molten globule state. A model derived for the three-dimensional structure of the molecule indicates that the subunits exist in two distinct conformations in the tetramer, leading to different relative accessibilities of cysteine and tryptophan residues, as observed in solution.